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Dynamic evolution of Geranium mitochondrial genomes through multiple horizontal and intracellular gene transfers Seongjun Park1, Felix Grewe2, Andan Zhu2, Tracey A. Ruhlman1, Jamal Sabir3, Jeffrey P. Mower2 and Robert K. Jansen1,3 1

Department of Integrative Biology, University of Texas, Austin, TX 78712, USA; 2Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA;

3

Department of Biological Science, Biotechnology Research Group, King Abdulaziz University, Jeddah 21589, Saudi Arabia

Summary Author for correspondence: Robert K. Jansen Tel: +1 512 471 8827 Email: [email protected] Received: 15 December 2014 Accepted: 15 April 2015

New Phytologist (2015) 208: 570–583 doi: 10.1111/nph.13467

Key words: cox1 intron, gene conversion, Geraniaceae, homing endonuclease, multiple organellar genes, parasitic plant.


The exchange of genetic material between cellular organelles through intracellular gene transfer (IGT) or between species by horizontal gene transfer (HGT) has played an important role in plant mitochondrial genome evolution. The mitochondrial genomes of Geraniaceae display a number of unusual phenomena including highly accelerated rates of synonymous substitutions, extensive gene loss and reduction in RNA editing.
Mitochondrial DNA sequences assembled for 17 species of Geranium revealed substantial reduction in gene and intron content relative to the ancestor of the Geranium lineage. Comparative analyses of nuclear transcriptome data suggest that a number of these sequences have been functionally relocated to the nucleus via IGT.
Evidence for rampant HGT was detected in several Geranium species containing foreign organellar DNA from diverse eudicots, including many transfers from parasitic plants. One lineage has experienced multiple, independent HGT episodes, many of which occurred within the past 5.5 Myr.
Both duplicative and recapture HGT were documented in Geranium lineages. The mitochondrial genome of Geranium brycei contains at least four independent HGT tracts that are absent in its nearest relative. Furthermore, G. brycei mitochondria carry two copies of the cox1 gene that differ in intron content, providing insight into contrasting hypotheses on cox1 intron evolution.

Introduction Horizontal gene transfer (HGT), or lateral gene transfer, is the movement of a segment of genetic material from one species into the genome of an unrelated species. HGT is prevalent across prokaryotes, but also occurs between prokaryotes and their eukaryotic hosts (Gogarten et al., 2002; Jain et al., 2002; Boucher et al., 2003) and among eukaryotes (Andersson, 2005; Keeling & Palmer, 2008). The implications of HGT in genome evolution have been studied extensively across land plants, especially in angiosperms (Richardson & Palmer, 2007; Bock, 2010). This phenomenon is common in plant mitochondrial genomes but few cases of HGT have been documented in the nucleus (Ghatnekar et al., 2006; Yoshida et al., 2010; Xi et al., 2012; Zhang et al., 2013b; Li et al., 2014). Plant mitochondrial genomes contain so-called promiscuous sequences because they have varying amounts of DNA from plastids (mitochondrial DNA of plastid origin, MIPTs), the nucleus (mitochondrial DNA of nuclear origin, MINCs) or even genetic material from other species via HGT (Mower et al., 2012a,b). HGT plays an important role in plant mitochondrial genome evolution. Many examples of HGT of one or multiple 570 New Phytologist (2015) 208: 570–583 www.newphytologist.com

genes into mitochondria have been documented in asterids (Bergthorsson et al., 2003; Mower et al., 2004; Sch€onenberger et al., 2005), rosids (Bergthorsson et al., 2003; Nickrent et al., 2004; Woloszynska et al., 2004; Xi et al., 2013), Papaveraceae (Bergthorsson et al., 2003), Magnoliaceae (Hepburn et al., 2012), Amborella (Bergthorsson et al., 2003, 2004; Rice et al., 2013) and the fern Botrychium (Davis et al., 2005). Group I or II introns that act as mobile genetic elements can also be transferred by HGT into mitochondrial genomes. For example, the cox1 group I intron was transferred from a fungus into angiosperms, and subsequent evolution within angiosperms has been characterized (Cho et al., 1998; Cusimano et al., 2008; Sanchez-Puerta et al., 2008). Another example of horizontal intron transfer involves the nad1i77 group II intron in Gnetaceae (Won & Renner, 2003), Rafflesiaceae (Davis & Wurdack, 2004) and Botrychium (Davis et al., 2005). The mitochondrial genome of the basal angiosperm Amborella exhibits massive horizontal transfer of multiple genes, even entire genomes, from mosses, green algae and other angiosperms (Rice et al., 2013). Horizontal acquisition of one or more genes can result in the duplication of genes in the recipient species (duplicative HGT) or recapture of genes that were previously lost as a consequence of intracellular gene transfer (IGT) Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

New Phytologist (recapture HGT). Possible mediators of HGT have been proposed, including direct exchange involving parasitic plants through haustoria (Davis & Wurdack, 2004; Mower et al., 2004, 2010; Xi et al., 2013), grafting (Stegemann et al., 2012; Fuentes et al., 2014) and transfer from epiphytes (Bergthorsson et al., 2004; Rice et al., 2013). Several additional vectors have been proposed, including pollen, fungi, bacteria, viruses and insects (Bergthorsson et al., 2003; Bock, 2010). The horizontal transfer of nucleotide sequences can be either DNA- (Mower et al., 2010) or RNA-mediated (Kim et al., 2014). Understanding the evolutionary mechanisms of HGT should provide new insights into developing a plant mitochondrial transformation system (Mileshina et al., 2011). Horizontal gene transfer provides a crucial source of genetic variation in mitochondrial genes, many of which display intron gain/loss and chimeric genes created by gene conversion. The angiosperm acquisition of the cox1 group I intron by horizontal transfer from fungi exhibits the hallmarks of HGT, including sporadic distribution across angiosperms, the presence of the coconversion tract (CCT; flanking exon sequences that are converted to donor DNA sequences) and phylogenetic incongruence (reviewed in Mower et al., 2012a). The double-strand break (DSB) repair pathway has been proposed to facilitate intron integration through the CCT of the foreign copy at a homing site (a target site) in the recipient intronless cox1 (Delahodde et al., 1989; Lambowitz & Belfort, 1993; Belfort & Perlman, 1995). HGT followed by gene conversion between the foreign and native cox2 copies has been proposed as a mechanism of intron loss in Magnolia mitochondrial DNA (Hepburn et al., 2012). Novel chimeric gene structures often arise from HGT and subsequent gene conversion between foreign and native sequences in angiosperm mitochondrial genomes (Barkman et al., 2007; Hao et al., 2010; Mower et al., 2010; Hepburn et al., 2012). Contributing to the complex evolutionary history of plant mitochondrial genomes, genetic material of mitochondria, including genes, can also be transferred to plastid (plastid DNA of mitochondrial origin, PLMTs; Mower et al., 2012a) or nuclear genomes (nuclear DNA of mitochondrial origin, NUMTs; Timmis et al., 2004) via IGT. NUMTs are common among angiosperms (Timmis et al., 2004) often resulting in loss-of-function of the mitochondrial-encoded copy (Adams et al., 2002b), whereas PLMTs are rarely observed (Iorizzo et al., 2012; Straub et al., 2013; Downie & Jansen, 2015). In Geraniaceae, mitochondrial genomes show evidence of a number of unusual evolutionary phenomena including highly accelerated rates of synonymous substitutions (especially in Pelargonium), extensive loss of genes (Erodium) and reduction in RNA editing (Pelargonium) (Adams et al., 2002b; Parkinson et al., 2005; Zhang et al., 2013a), none of which has been examined in Geranium, the largest genus in the family. To gain insight into the evolution of Geranium mitochondrial genomes, the genome sequences of 17 species of Geranium and eight related species in Geraniales were assembled. Geranium mitochondrial DNAs have experienced rampant, lineage- or species-specific HGT from diverse donors. The origin of foreign DNA and the Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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timing of HGT events are evaluated. The number of HGT events, the evolutionary fate of the transferred genes and the potential mechanisms that underlie these events are discussed. In addition, extensive losses of genes/introns and IGT to the nucleus in Geranium and related species are characterized.

Materials and Methods Taxon sampling and DNA/RNA sequencing Total genomic DNA was isolated from fresh leaf tissue of 17 species of Geranium plus Erodium texanum, Monsonia emarginata and Pelargonium cotyledonis as described previously (Park et al., 2014). Approximately 6 Gb of 100-bp paired-end reads were generated from an 800-bp insert library using the Illumina HiSeq2000 sequencing platform at the Genome and Sequence Analysis Facility (GSAF) at the University of Texas at Austin. Previously sequenced paired-end reads (Weng et al., 2014) for California macrophylla and Hypseocharis bilobata from Geraniaceae and Francoa sonchifolia (Francoaceae), Melianthus villosus (Melianthaceae) and Viviania marifolia (Vivianiaceae) were also used. To evaluate potential HGT sources, four parasitic plant taxa were included: Rafflesia cantleyi (SRR629613) and Sapria himalayana (SRR629601) from Malpighiales (Xi et al., 2013), Cuscuta gronovii from Solanales, and Bartsia pecularoides from Lamiales. Genomic DNA for Cuscuta gronovii was provided by Sasa Stefanovic (University of Toronto Mississauga). Bartsia genomic DNA was isolated from silica-dried leaves with the Plant DNeasy Kit (Qiagen). Cuscuta gronovii and Bartsia DNAs were sequenced by BGI, generating c. 6 Gb of 100-bp paired-end reads from an 800-bp library using the Illumina HiSeq2000 platform. Total RNA isolations from three Geranium species (G. incanum, G. maderense and G. phaeum) and three outgroups (C. macrophylla, E. texanum and M. emarginata) were performed as described by Zhang et al. (2013a) and Illumina RNAseq library construction and sequencing were carried out at the GSAF. Genome assembly and gene annotation In order to identify mitochondrial genes from each genome, paired-end Illumina reads were assembled de novo with Velvet v1.2.08 (Zerbino & Birney, 2008) at the Texas Advanced Computing Center (TACC) using multiple k-mers (61–91) and expected coverage values (100–500) with coverage cut-off set to 10% of the expected coverage. The initial mitochondrial contigs from G. brycei were assembled manually in Geneious R6 v6.1.8 (Biomatters Ltd, Auckland, New Zealand, http://www.geneious.com/) and a nearly complete draft mitochondrial genome was annotated using MITOFY (Alverson et al., 2010). For G. maderense, a complete mitochondrial genome was assembled from the initial velvet contigs and then annotated as described previously (Grewe et al., 2014; Zhu et al., 2014). For the other species, mitochondrial genes were identified in each draft assembly by BlastN using protein-coding genes from G. brycei and related rosids as query sequences. Mitochondrial gene sequences were deposited in GenBank (Supporting Information Table S1). New Phytologist (2015) 208: 570–583 www.newphytologist.com

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Intron nomenclature for mitochondrial genes follows Dombrovska & Qiu (2004). RNA editing sites were predicted by PREP-Mt (Mower, 2009) with a cutoff value of 0.5. Identification of IGT Transcriptomes from three Geranium species (G. incanum, G. maderense and G. phaeum) and three outgroups (C. macrophylla, E. texanum and M. emarginata) were assembled de novo with Trinity (Grabherr et al., 2011; released on 25 February 2013) using the script from Zhang et al. (2013a) at TACC. Nuclearencoded copies were identified in each assembly by using BlastN (e-value cutoff of 1e-10) with organelle protein-coding genes from related rosids as query sequences. Mitochondrial targeting peptides (mTP) were predicted by MitoprotII v1.101 (Claros & Vincens, 1996), Predotar v1.03 (Small et al., 2004) and TargetP v1.1 (Emanuelsson et al., 2007). Detection and evaluation of HGT In order to evaluate the possibility that putative HGT events could be DNA contamination or a nuclear gene copy, the depth of coverage of native vs foreign regions was compared using Bowtie2 v2.1.0 (Langmead & Salzberg, 2012) on TACC. For phylogenetic analyses of HGT, sequenced mitochondrial and plastid genes across angiosperms were selected (Tables S2, S3). The datasets for individual mitochondrial and plastid genes were aligned with MUSCLE (Edgar, 2004) in Geneious R6. Phylogenetic trees were constructed using maximum-likelihood methods in RAxML v7.2.8 (Stamatakis, 2006) with the ‘GTRGAMMA’ model under the rapid bootstrap algorithm (1000 replicates) on TACC. Gene conversion was detected by GENECONV in the Recombination Detection Program (RDP) v4.36b (Martin et al., 2010). Estimation of divergence time Divergence times were estimated by Bayesian MCMC methods using BEAST v2.1.3 (Bouckaert et al., 2014). Five plastid markers (psbA, psbB, psbC, psbD and rbcL) were chosen to estimate divergence times among Geraniales (Table S4). The GTR + I + G substitution model was selected based on the corrected Akaike information criterion in jModeltest 2.1.5 (Darriba et al., 2012). A relaxed clock with lognormal distribution of uncorrelated rate variation was specified and a Yule speciation model was used to model the tree prior. For calibration points, one fossil as minimum age constraint (mean = 0.0, SD = 0.75, offset = 28.4) was used with a lognormal prior distribution (the fossil for Geraniaceae excluding Hypseocharis; 28.4  0.1 Myr ago (Ma), Palazzesi et al., 2012) and four additional points as the root age constraint were used with a normal prior distribution. The four calibration points were chosen based on previously published divergence time estimates of angiosperms (Bell et al., 2010): angiosperms (mean = 183, SD = 10, range 167–199 Ma), eudicots (mean = 130, SD = 4, range 123–139 Ma), rosids (mean = 125, SD = 4, range 118–132 Ma), and asterids (mean = 110, SD = 5.5, range New Phytologist (2015) 208: 570–583 www.newphytologist.com

101–119 Ma). Analysis under each set of constraints was run for 200 000 000 generations (trees sampled at every 1000 generations). The sufficient effective sample size (larger than 200) and convergence of the Markov chain Monte Carlo chains were determined in Tracer v1.6 (Rambaut et al., 2014) with 10% burn-in.

Results Reduction in native mitochondrial gene content due to IGT Mitochondrial contigs were assembled from paired-end Illumina reads for 17 Geranium species and eight related species in Geraniales. The average coverage of draft genomes ranged from 58 9 to 376 9 (Table S5). Using these contigs from the draft assembly, a complete mitochondrial genome of 737 kb was assembled for G. maderense (Fig. S1, accession number KP940515). In addition, a high-quality draft genome for G. brycei was assembled into eight scaffolds totaling 1053 kb in length, although the complete genome is likely to be larger due to the presence of repetitive DNA (Fig. 1, accession numbers KP974311–KP974318). All Geranium mitochondrial DNAs contain 26 or 27 protein-coding genes with five cis-spliced and four trans-spliced introns that are most similar to gene and intron sequences from the other Geraniales species (Figs S2, S3), suggesting that these are native copies inherited vertically from the Geranium common ancestor. With one exception (loss of rps3), losses of protein-coding genes and introns are shared by all examined Geranium species (Figs S2, S3). PREP-Mt predicted fewer RNA editing sites in Geranium than in Arabidopsis (Table S6). Start codon editing (ACG to AUG) was predicted for the nad1 gene and an alternative start codon (ATA) was noted for the mttB gene in all species of Geranium examined. Available transcriptome data confirmed the edited start codon for the G. incanum, G. maderense and G. phaeum nad1 genes. Multiple gene transfers from the mitochondria to the nucleus were detected in the Geraniaceae transcriptome data (Table S7). Acquisition of mitochondrial targeting peptides in nearly all predicted open reading frames (ORFs) of the transcripts suggests their functionality. Many predicted ORFs reflect intact mitochondrial genes, whereas the predicted ORFs of rpl2 that are present as two sequentially acquired ORFs lack a mitochondrial copy (Fig. S4; Table S7). Two different transcripts were detected for rpl5 and rps14 in G. maderense and for rps19 and sdh3 in G. phaeum (Table S7). Transcripts for 50 rpl2, 30 rpl2, and rps19 were also found in four additional transcriptomes from other genera in Geraniales (i.e. Francoa sonchifolia, KP963188–KP963190; Hypseocharis bilobata, KP963185–KP963187; Melianthus villosus, KP963182–KP963184; Pelargonium cotyledonis, KP963179– KP963181), suggesting that the IGT events occurred in the common ancestor of Geraniaceae or Geraniales. Gain of multiple foreign genes by HGT The mitochondrial genome of several Geranium species contains additional mitochondrial gene copies that are either not typically Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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100 000

200 000

300 000

400 000

500 000

600 000

700 000

800 000

psbD (pt)

900 000

1000 000

10 kb

Fig. 1 Draft of the 1053-kb Geranium brycei mitochondrial genome. The eight contigs are placed in linear order with triangles indicating the boundaries of each contig. Genes above and below each contig indicate the direction of transcription. Genes in blue are native copies; foreign copies of mitochondrial genes inferred from phylogenetic analyses are color-coded by potential donors (red, Euphorbiaceae, Malpighiales; orange, Cuscuta from Convolvulaceae, Solanales; black, unidentified donor from eudicots; gray, unidentified donor from Rubiaceae, Gentianales). pt, genes of plastid origin.

found in Geranium species or are more similar to other angiosperm homologs. GC content and predicted RNA editing sites differ between the typical Geranium genes and the additional gene copies (Table S8), indicating that the additional copies were not derived by duplication or retroprocessing of Geranium genes. Several observations rule out the possibility that these additional gene copies are due to DNA contamination. First, the depth of coverage of these genes was very similar to other contigs of Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

mitochondrial DNA (Table S9), whereas a contaminant would be expected to be present at a much lower copy number in the DNA sample. Second, 13 of the 29 extra mitochondrial genes have a frameshift mutation or are degraded (Table 1), whereas most to all contaminant sequences should appear intact and functional. Finally, some phylogenetic trees (atp1, atp6, cox3, matR) contain several native genes that lack additional copies (Fig. S5); because these four genes are present in the mitochondrial New Phytologist (2015) 208: 570–583 www.newphytologist.com

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574 Research Table 1 Horizontal gene/DNA transfers into the mitochondrial DNAs of Geranium Genes Mitochondrial atp4 atp8 ccmC ccmFc (ccmFci829) cob cox1 (cox1i729) cox2 (cox2i373) mttB nad2 exon4,5 (nad2i1282) nad6 rpl5 rpl16 rps1 rps3 (rps3i74) rps4

rps10 (rps10i235) rps14 Plastid rbcL psaA psaB atpB atpE rps14 rpoB 30 rpoC1 exon1 petB rpl2 intron psbD ndhJ matK

Recipients

Potential HGT donors

HGT events

Gene integrity

G. brycei G. incanum G. brycei G. brycei G. incanum G. brycei G. brycei G. incanum G. brycei G. incanum G. incanum G. brycei G. traversii G. brycei G. brycei G. incanum G. brycei G. brycei G. incanum G. brycei G. brycei* G. endressii G. incanum* G. nodosum G. platyanthum G. sanguineum G. traversii G. brycei G. brycei

Gentianales eudicots

Duplicate Duplicate Duplicate Duplicate Duplicate (recapture) Duplicate Duplicate Duplicate (recapture) Duplicate (recapture) Duplicate Duplicate (recapture) Duplicate (recapture) Duplicate Recapture Recapture Duplicate Duplicate Duplicate (recapture) Recapture Recapture Recapture Recapture recapture Recapture Recapture Recapture Recapture Recapture Recapture

I I I I w I w I I I IP IP I I I I I w w w w w w w w w w I w

Solanales/Convolvulaceae/Cuscuta Malpighiales/Euphorbiaceae/Acalyphoideae Solanales/Convolvulaceae/Cuscuta Malpighiales/Euphorbiaceae Solanales/Convolvulaceae/Cuscuta Solanales/Convolvulaceae/Cuscuta Solanales/Convolvulaceae/Cuscuta Lamiales/Orobanchaceae Solanales/Convolvulaceae/Cuscuta Malpighiales/Euphorbiaceae/Acalyphoideae Malpighiales/Euphorbiaceae/Acalyphoideae Malpighiales/Euphorbiaceae/Acalyphoideae Malpighiales/Euphorbiaceae/Acalyphoideae

Solanales/Convolvulaceae/Cuscuta

Malpighiales/Euphorbiaceae/Acalyphoideae Solanales/Convolvulaceae/Cuscuta

G. brycei G. incanum

Solanales/Convolvulaceae/Cuscuta

G. brycei G. incanum

Malpighiales/Euphorbiaceae/Acalyphoideae

G. brycei G. incanum

Gentianales/Rubiaceae/Rubioideae

w w w w w w/I w w I w w w w

* Copies of rps4 from G. brycei (295 bp) and G. incanum (575 bp) were not used in the phylogenetic analyses due to alignment problems, but these two gene sequences are more similar to Cuscuta rps4 than other Geranium species. In the case of plastid rps14, G. brycei is a pseudogene and G. incanum is an intact gene. I, intact gene; P, partial; w, pseudogene.

genomes of all angiosperms sequenced to date, they should have been recovered from a contaminant. Taken together, these factors indicate that the additional gene copies are foreign DNA sequences acquired by HGT. These events represent duplicative HGT (atp4, atp8, ccmC, ccmFc, cob, cox1, cox2, mttB, nad2 exon4/5, nad6, rps1 and rps3) and recapture HGT (rpl5, rpl16, rps4, rps10 and rps14) in Geranium species. Some of the transferred mitochondrial genes are intact, suggesting potential functionality (Table 1). In addition to foreign mitochondrial gene copies, 13 foreign angiosperm MIPTs are present as intact genes New Phytologist (2015) 208: 570–583 www.newphytologist.com

(2) or pseudogenes (12) in both G. brycei and G. incanum (Table 1). Phylogenetic analyses indicated the origin of foreign gene sequences (Figs 2a–f, S6). Several Geranium species acquired multiple mitochondrial genes from different donors representing four orders of angiosperms (Gentianales, Lamiales, Malpighiales and Solanales) and a single gene from one unidentified eudicot. About half of these horizontally acquired genes appear to have originated from parasitic plants due to their strong association with either Cuscuta, the sole parasitic genus in Convolvulaceae Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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(a) cox2

51 61 89

G. phaeum G. bryceiψ 100 (c) nad6 G. endressii G. incanumψ G. traversii G. nodosumψ G. nodosum G. endressii 100 50 62 99 G. brycei G. traversii G. incanum 76 G. platypetalum G. platypetalum G. phaeum 53 G.maderense G. maderense 99 100 81 100 G. macrorrhizum G. macrorrhizum Erodium 75 100 Erodium 100 California Monsonia 96 72 Monsonia Hypseocharis Hypseocharis Viviania Cucurbita Salvia 100 Asclepias Bartsia 56 Rhazya Asclepias 100 99 G. traversii Rhazya Bartsia Nicotiana 53 94 Salvia Cuscuta 88 92 Mimulus Beta Boea Daucus Daucus Helianthus Helianthus 61 Vaccinium Nicotiana 89 98 Ricinus Cuscuta 81 G. bryceiψ Beta 99 Hevea Arabidopsis Rafflesia 100 Vaccinium Sapria 80 Francoa 100 Batis 97 Melianthus 98 Arabidopsis Viviania Carica 96 Sapria 99 Gossypium 62 Rafflesia Glycine 73 100 93 Ricinus Vigna Hevea 84 Malus Glycine Cucurbita 78 99 58 Vigna Melianthus Carica 98 Francoa Gossypium 96 Vitis Batis Phoenix 86 56 Malus 99 Oryza Vitis Spirodela Liriodendron Liriodendron Phoenix 64 Amborella Oryza 66 Spirodela Amborella

100 G. incanum

(b) rps3

G. brycei G. nodosum

98 G. endressii

G. traversii G. platypetalum G. phaeum 89 G. maderense G. macrorrhizum Monsonia 80Erodium California Hypseocharis

63 100 92

Rhazya Asclepias Beta Bartsia 100 Salvia G. brycei 100 G. incanum Cuscuta 88 Nicotiana Arabidopsis 84 Batis 55 Carica Gossypium Ricinus 94 66 Hevea Sapria 100 Rafflesia Melianthus Viviania 91 Francoa Cucurbita Glycine Malus 100 Helianthus Daucus 56 Vaccinium Vitis Liriodendron Phoenix 55 75 Oryza Spirodela Amborella 98

G. traversii G. nodosum

(d) mttB

(e) rps4

96 G. endressii 63 G. platypetalum G. incanum 87 69 G. brycei G. maderense 63

100

61 68

62

98 56

G. phaeum G. macrorrhizum 57 Erodium Monsonia California Hypseocharis

74

100

69

51

Daucus Rhazya Asclepias Helianthus Cuscuta 99 G. brycei 97 Ricinus 92 Hevea 86

79

100

Melianthus

Sapria G. incanumψ G. bryceiψ

98 Francoa 67

Vitis

Arabidopsis Carica Gossypium Francoa Melianthus 92 Cucurbita 100 Glycine Vigna Phoenix 100 72 Oryza Spirodela 54 Liriodendron Amborella

100 98

Cuscuta 69 50

91

C. africana C. angulata C. nitida

80

100

80 53

98

Malus Cucurbita Vigna Glycine Batis 81 Arabidopsis Carica Gossypium Vitis Oryza Phoenix Spirodela

93

Liriodendron Amborella

(h) rpoB 3' & rpoC1 exon1

C. gronovii C. yucatana C. hyalina ψ 100 G. brycei 100 G. incanumψ C. planiflora C. cassytoides C. reflexa

Erycibe Ipomoea Merremia Convolvulus 100 Calystegia 86 Falkia Neuropeltis 57 99 S. lycopersicum 74 S. tuberosum 100 Nicotiana Atropa 52

99

Rafflesia

Sapria Beta

Rafflesia

54

rbcL

Salvia Bartsia Vaccinium

98

Ricinus

100

Convolvulaceae

(f) rpl5

G. endressiiψ Cuscuta

100

Ricinus 79 Hevea 100 Glycine Vigna Malus Viviania Beta Francoa Melianthus 100 G. incanum 56 74 Cuscuta Nicotiana Salvia 82 100 Bartsia Asclepias 64 98 Rhazya Daucus Vaccinium 86 Helianthus Vitis (g) 92 Phoenix Oryza Spirodela 54 Liriodendron Amborella

ψ

Nicotiana Asclepias Rhazya Bartsia 100 Salvia Helianthus Daucus Vaccinium Beta

50

Sapria Rafflesia

99

88 G. platyanthum 64 G. traversiiψ 82 G. nodosumψ 100 G. sanguineumψ

94

Arabidopsis Batis Carica Cucurbita Gossypium 100

82 51

100

Solanales

Euphorbiaceae

G. bryceiψ G. incanumψ Ricinus Hevea 98 98 Manihot Malpighiales Jatropha 98 74 Couepia 100 Licania Parinari 89 Populus 100

100

75

0.01 substitutions per site

Fig. 2 Phylogenetic evidence for horizontal transfer of multiple genes into Geranium mitochondrial genomes. Maximum-likelihood trees based on (a–f) mitochondrial and (g, h) plastid genes. Bootstrap support values > 50% are shown on the branches. Colors indicate native (blue) and foreign copies corresponding to multiple potential donors (red, Euphorbiaceae, Malpighiales; orange, Cuscuta from Convolvulaceae, Solanales; purple, Orobanchaceae, Lamiales). All trees were drawn to the same scale. Bold branches correspond to (d, e, g) 0.02, (a) 0.04 or (b) 0.08 substitutions per site. See Supporting Information Figs S5 and S6 for additional evidence for horizontal gene transfer of the other genes or more species-rich analyses. Ψ, pseudogene.

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(Solanales), or Bartsia, one of many parasitic members of the Orobanchaceae (Lamiales). The two copies of rps4 found in both G. brycei and G. incanum appear to have been recaptured from different donors (Table 1). Phylogenetic analyses of the foreignlike MIPTs allow more precise determination of potential donors due to the increased sampling, providing strong support for Cuscuta within Convolvulaceae, for a member of Acalyphoideae (represented by Ricinus) in Euphorbiaceae (Malpighiales) and for a member of Rubioideae (represented by Phyllis) in Rubiaceae (Gentianales) (Figs 2g,h, S7). The pattern of gene clustering on the G. brycei mitochondrial genome (Fig. 1) provides strong support for several results presented earlier from phylogenetic analyses, GC content and RNA editing. The clustering of genes from the same donor provides strong evidence for HGT rather than phylogenetic artifact because artifacts should be random. The clustering also indicates that the transfers were DNA mediated. The clustering of transferred plastid and mitochondrial genes from the same donor (e.g. rps10, cox1, petB and rps1 in red in Fig. 1) clearly indicates that the plastid gene (petB) was transferred from the mitochondrial genome of the donor, and not from the plastid genome of the donor. The clustering also provides more confidence in the identification of the donor for each gene in the cluster, even in cases when the phylogenetic results do not provide strong support for a particular gene. For example, only one gene tree (rpoB/rpoC1, Fig. S7g) provided strong bootstrap support for the Ricinus lineage transfer, whereas three gene trees (rpl2 (Fig. S7i), ndhJ (Fig.

S7k) and rps4 (Fig. 2e)) had weak support. However, the clustering of these genes on the G. brycei mitochondrial genome supports the Ricinus lineage as the donor (Fig. 1). Gene conversion between foreign and native copies was detected for cox2 in G. brycei but not in G. incanum (Fig. 3a,b). Three alternative overlapping recombination regions of 58, 89, and 128 nt with significant support (P < 0.001) were identified (Fig. 3a,c). The three alternatives are highly similar (99.2–100% sequence identity) to the native copy, suggesting that the foreign copy has undergone gene conversion with the native G. brycei sequence (Fig. 3a,c). Horizontal transfer of mitochondrial DNA in Geranium includes group I and group II introns (i.e. cox1i729, ccmFci829, cox2i373, nad2i1282, rps3i74 and rps10i235; Table 1) that were previously lost in Geranium (Fig. S3), providing further support that the horizontal transfer of these sequences was DNA-mediated. The cox1 group I intron (cox1i729) with two truncated exons was found downstream of the foreign rps10 gene (Fig. 4a). Integration of the cox1i729 intron into the intronless cox1 gene of G. brycei was not observed (Fig. 4b). The transferred copy retains the intact homing endonuclease that resides in cox1i729 intron and very little (306 and 78 nt) of the adjacent exons (Fig. 4a). The remaining exon sequences provide diagnostic nucleotide substitutions, especially in the region indicated in Fig. 4(c) (CCT); additional differences (positions + 27, + 36, + 39 and + 69) were observed downstream of the 30 CCT (Fig. 4c). A homoplasious site (position 25), possibly a 50 CCT,

(a)

(b)

(c)

Fig. 3 Gene conversion between the native and foreign copies of cox2. (a) Nucleotide positions of three significant recombination regions are outlined in boxes. The blue box indicates the most likely recombination region involved in the formation of a chimeric sequence. Colored bars on the alignment map indicate nucleotide sites that differ from the consensus sequence (red, A; blue, C; yellow, G; green, T). (b) Model of horizontal gene transfer (HGT) of an intron-containing cox2 gene followed by gene conversion with native copy in Geranium brycei. An inferred HGT event occurred in the common ancestor of G. brycei and G. incanum, then both species acquired the intron-containing cox2 via vertical gene transfer (VGT). Colored boxes indicate native (blue) and foreign (pink) cox2 genes. Lines joining two exons of the foreign copy indicate the intron. (c) Nucleotide alignment of the 128-nt region from (a) showing gene conversion between the foreign and native copy of G. brycei. Boxes with arrows indicate the three alternative recombination regions from (a). The number ‘2’ after G. brycei and G. incanum denotes foreign copies. New Phytologist (2015) 208: 570–583 www.newphytologist.com

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was detected upstream of the intron. The arrangement of the rps10 sequences upstream of the cox1 gene in G. brycei is more similar to Ricinus in Euphorbiaceae than to the Hevea sequence (Fig. 4a). The cox1i729 intron of G. brycei grouped with Acalypha, another member of the Euphorbiaceae (Fig. S6e). Both results strongly suggest that the donor was a member of Acalyphoideae, of which both Ricinus and Acalypha are members. Timing of evolutionary events The most parsimonious scenario for gene transfer events (IGT and HGT) and divergence time estimates for major nodes within Geraniales is summarized in Fig. 5 (see Fig. S8 for more detailed divergence time estimates). The IGTs of 50 rpl2 and rps19 occurred 95 Ma in Geraniales after the more ancient IGT of 30 rpl2 (125 Ma). Divergence time estimates suggest that extensive IGT events occurred in Geraniaceae c. 61 Ma (95% highest posterior density = 40–83 Ma). A subsequent IGT event involving rps12 occurred 35 Ma in Geraniaceae excluding Hypseocharis. The IGTs of the rps3 gene are recent (1.0 and 3.6 Ma) and restricted to two Geranium clades (Fig. 5). The transfer of the rps4 gene from Cuscuta into the common ancestor of eight Geranium species occurred

(a)

5.5 Ma with subsequent HGT events occurring in different Geranium clades. For example, HGT of nad6 from Orobanchaceae into G. traversii occurred ≤ 3.7 Ma (Fig. 5). The HGT of five genes (atp8, cox2, nad2, rps1 and rps4) from Cuscuta, Acalyphoideae or an unidentified eudicot occurred in the common ancestor of the G. brycei and G. incanum clade (1.0–5.5 Ma). Many subsequent, independent HGT events (atp4, ccmC, ccmFc, cob, cox1, mttB, rpl5, rpl16, rps14, rps3 and rps10) from Cuscuta, Acalyphoideae or an unidentified Rubiaceae (Rubioideae) occurred in G. brycei and G. incanum (Fig. 5).

Discussion In plant cells, HGT is much more common in mitochondrial genomes than in their nuclear or plastid counterparts (Keeling & Palmer, 2008). This is because plant mitochondria are generally more permissive in the uptake of foreign DNA and more active in mitochondrial fusion and fission (Richardson & Palmer, 2007; Bock, 2010). Geranium mitochondrial DNAs have experienced rampant HGT within specific lineages. The presence of foreign DNAs in Geranium mitochondrial genomes is the most extensive of any eudicot examined

cox1 exon1

cox1 exon2

Hevea rps10 exon1

rps10 exon2

Ricinus G. brycei rps10 exon1

rps10 exon2

306 nt

Homing endonuclease

78 nt

300 bp

(b) Ancestral Intron-containing cox1 HGT Homing endonuclease

(c)

–30

–20

–10

–1

+1

+10

+20

+30

+40

+50

+60

+70

Sequence Logo

lntronless cox1 Present

Hypothetical Endonuclease

DSB

Malpighia Hura Hevea Euphorbia Croton Rafflesia Acalypha Ricinus G. brycei2 G. brycei G. nodosum G. endressii G. incanum G. phaeum G. platypetalum G. traversii G. maderense G. macrorrhizum

+ C A C C C T GA A G T T T A C A T C C T A A T T C T GC C T GGA T T C GG T A T C A T A A G T C A T A T C G T T T C GA C T T T T T C GG + C A C C C T GA A G T T T A T A T T C T C A T T C T GC C GGGA T C C GG T A T C A T A A G T C A T A T C G T T T C GA C T T T T T C GG T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T + C A C C C T GA A G T T T A C A T T C T A A T T C T GC C GGGA T C C GG T A T C A T A A G T C A T A T C G T T T C GA C T T T T T C GG T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T + C A C C C T GA A G T T T A C A T T C T A A T T C T GC C T GGA T C C GG T A T C A T A A G T C A T A T C G T T T C T A C T T T T T C GG T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T + C A C C C T GA A G T T T A C A T C C T A A T T C T GC C T GGA T C C GG T A T C A T A A G T C A T A T C G T T T C T A C T T T T T C GG T T A T A C C A GC A T C T T T T T T GG T T C T T C GG T + C A C C C T GA A G T T T A C A T C C T A A T T C T GC C T GGA T C C GG T A T C A T A A G T C A T A T C G T T T C GA C T T T T T C GG T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T + C A C C C T GA A G T T T A C A T C C T A A T T C T GC C T GGA T C C GG T A T C A T A A G T C A T A T C G T T T C GA C T T T T T C GG T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T + C A C C C T GA A G T T T A C A T C C T A A T T C T GC C T GGA T C C GG T A T C A T A A G T C A T A T C G T T T C GA C T T T T T C GG T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T + C A C C C GGA A G T T T A C A T C C T A A T T C T GC C T GGA T C C GG T A T C A T A A G T C A T A T C G T T T C T A C T T T T T C GG T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C C GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A T C A GC A T C T C T T T T GG T T C T T C GG T - C A T C C A GA GG T C T A T A T T C T A A T T C T C C C T GGA T T T GGGA T C A T A A G T C A T A T C G T T T C C A C T T T T T C A G T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T T T A T A C C A GC A T C T C T T T T GG T T C T T C GG T

Coconversion tract

Fig. 4 Horizontal transfer of the group I intron of the cox1 gene in Geranium brycei with coconversion of flanking exons. (a) Schematic diagram of the genomic regions surrounding the intron-containing cox1 from G. brycei mitochondrial genome compared to regions from potential donors. Pink boxes indicate foreign rps10 and cox1 copies of G. brycei; white boxes indicate rps10 or cox1 genes of Ricinus and Hevea from Euphorbiaceae. Exon and introns were drawn to the same scale. (b) Model of horizontal gene transfer (HGT) of an intron-containing cox1 gene and subsequent truncation of two exons of foreign copy. Colored boxes indicate native (blue) and foreign (pink) cox1 genes. Lines joining two exons of foreign copy indicate the intron. The hypothetical model illustrates the potential insertion of a group I intron from the foreign intron-containing cox1 gene into the native copy through a double-stranded break repair pathway (DSB) after the activity of homing endonucleases. (c) Nucleotide alignment of cox1 exonic regions immediately flanking the intron insertion site. The sequence logo shows the most conserved bases represent coconversion tract (CCT) diagnostic signals that are not conserved at third-position synonymous sites. Symbols between the exons indicate cox1 intron presence (+) or absence (). The number ‘2’ after G. brycei denotes foreign copy. Arrow, well-known 30 CCT motif (Cho et al., 1998; Sanchez-Puerta et al., 2008). Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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Horizontal gene transfer (HGT) Intracellular gene transfer (IGT)

atp4

?

Asterids

nad6

rps4 mttB

nad2 ccmC

cox2 rps3

cob rpl5 rps14

3.6 3.7 5.5

3’ rpl2

Eudicots

1.0

rps3

125

rpl5, rpl16, rps4, rps7, rps10, rps13, rps14, sdh3, sdh4

rps2 rps11

rps12

cox1 rps10 rpl16 rps3

35 61

134 95

5’ rpl2, rps19

ccmFc

atp8 rps1

?

Rosids

rps4

Monocots

Fig. 5 Summary of horizontal gene transfer (HGT) and intracellular gene transfer (IGT) events in Geranium with divergence times. Colors correspond to potential donors (red, Euphorbiaceae, Malpighiales; orange, Cuscuta from Convolvulaceae, Solanales; purple, Orobanchaceae, Lamiales; gray, unidentified donors from Gentianales or eudicots). Species names and branches of Geranium are in blue. Numbers at nodes indicate divergence time estimates in Ma. See Supporting Information Fig. S7 for more complete divergence time estimates for the Geraniales. Gene names involved in HGT are shown in circles with lines and genes involved in IGT events are shown in boxes with dotted lines. IGT events in bold were confirmed using transcriptome data.

so far (except holoparasite Rafflesia; Xi et al., 2013). The mitochondrial genomes of Geranium and related genera of Geraniaceae also exhibit a substantial reduction in gene content due to intracellular transfer into the nucleus. These findings indicate that HGT and IGT have played a major role in shaping mitochondrial evolution in the family. New Phytologist (2015) 208: 570–583 www.newphytologist.com

Extensive mitochondrial gene loss and intracellular transfer to the nucleus Numerous Southern blot and genome sequencing surveys have shown that there is major variation in mitochondrial gene content among plants: some plants have lost most ribosomal protein Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

New Phytologist and succinate dehydrogenase genes, other plants have retained most of them in the mitochondrial genome, and the majority of plants fall somewhere in between (Mower et al., 2012b). For example, Liriodendron and Vitis have retained a nearly full complement of mitochondrial genes inherited from the angiosperm common ancestor (Goremykin et al., 2009; Richardson et al., 2013), whereas other plants, such as Silene and Ajuga, have lost nearly all of their mitochondrion-encoded ribosomal protein and succinate dehydrogenase genes (Sloan et al., 2012; Zhu et al., 2014). Several studies have shown that these missing mitochondrial genes were relocated to the nucleus (e.g. Adams et al., 2002b; Liu et al., 2009), although in some cases the mitochondrial genes were lost after functional replacement by plastidderived or cytosolic paralogs in the nuclear genome that were retargeted to the mitochondrion (e.g. Adams et al., 2002a; Mollier et al., 2002; Mower & Bonen, 2009). Extensive loss of protein-coding genes and introns was observed in all examined Geranium mitochondrial genomes. As in other angiosperms, ribosomal and sdh genes were the most commonly affected (Adams et al., 2002b). The protein coding content of Geranium mitochondria includes only 26 or 27 of the 41 genes present in the common ancestor of angiosperms (Mower et al., 2012b). Comparative analyses of gene and intron content among mitochondrial genomes of other Geraniales suggest that two genes (rpl2 and rps19) are lost in the common ancestor of Geraniales and most gene losses (rpl5, rpl16, rps4, rps7, rps10, rps13, rps14, sdh3 and sdh4) occurred in the common ancestor of Geraniaceae, whereas two losses (rps12 and rps3) were unique to specific lineages within the family (Fig. 5). Transcriptomes of three Geranium species and the related genera California, Erodium and Monsonia provide evidence that the missing genes have been transferred to the nucleus, and that they have acquired transit peptides to target the products back to the mitochondrion. Previous studies have documented that the 30 portion of the rpl2 gene was transferred to the nucleus in the common ancestor of core eudicots (Adams et al., 2001; Adams & Palmer, 2003). Identification of both 50 rpl2 and 30 rpl2 that have each acquired target peptides in the transcriptomes indicates that the 50 rpl2 has been transferred to the nucleus in the common ancestor of Geraniales after the split of this gene between the mitochondrial and nuclear genomes in other eudicots (Fig. S4; Table S7). Several underlying reasons for extensive IGT in some lineages with low rates of transfer have been proposed (Martin & Herrmann, 1998; Blanchard & Lynch, 2000; Wade & Brandvain, 2009) but the relative contribution of selective and nonadaptive processes is unclear. In Geraniaceae and Silene, which have accelerated mitochondrial substitution rates (Parkinson et al., 2005; Mower et al., 2007; Sloan et al., 2012), selection could be driving the massive transfer of genes into the nucleus to avoid Muller’s ratchet. IGT could be adaptive for increased nuclear regulation of organellar processes. Alternatively, it may proceed more quickly in clonal and selfing plants due to the minimization of negative interactions between mitochondrial and nuclear factors (Brandvain et al., 2007). Or, the pattern of retention vs loss could largely be a function of the relative rates of mitochondrial DNA transfer to the nucleus, without any selective reason for it. Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

Research 579

Multiple horizontal transfers of organelle genes into Geranium mitochondria The Geranium mitochondrial genomes have received a considerable number of foreign organellar genes from diverse donors. The most recent HGT events occurred ≤ 5.5 Ma and are restricted to individual species or specific lineages, especially G. brycei and G. incanum (Fig. 5). The phylogenetic position of five of the foreign genes (atp8, cox2, nad2, rps1 and rps4) suggests that there were three independent HGT events in the common ancestor of G. brycei and G. incanum and that the transferred genes were thereafter vertically inherited by the two species. In addition, many subsequent horizontal acquisitions of single or multiple genes occurred via independent HGT in G. brycei and G. incanum (Fig. 5). Alternatively, some of the events could be explained by a single HGT in common ancestor of both species followed by independent losses. Horizontal gene transfers into mitochondria are classified into two major types (duplicative and recapture HGT) based on their evolutionary history and represent the various outcomes of HGT events: functional replacement of the native copy, loss of the foreign copy (silent) and generation of a chimeric gene through gene conversion with the native copy (Bergthorsson et al., 2003; Mower et al., 2012a). In Geranium, most foreign genes are present as duplications with five exceptions: rpl5, rpl16, rps4, rps10 and rps14. All examined Geranium species lack these genes in their mitochondrial genomes suggesting an ancient loss in the ancestor of Geraniaceae (Fig. 5). Thus, these five genes have been acquired by recapture HGT in Geranium. Two different copies of rps4 were detected in G. brycei and G. incanum mitochondrial genomes that likely arose through recurrent horizontal transfer events in their common ancestor. In both species the two mitochondrial copies of rps4 have experienced pseudogenization, and a putative functional copy was identified in G. incanum transcriptome (Table S7) suggesting that a functional IGT event had occurred permitting the loss of the mitochondrial copies. In addition to rps4, other horizontally acquired sequences are pseudogenes indicating nonfunctional or silent transfers (Table 1). Experimental evidence has shown that many intact genes of foreign origin are nonfunctional, although some foreign genes are transcribed (Mower et al., 2010; Rice et al., 2013). Xi et al. (2013) argued that most of the foreign genes in Rafflesia mitochondrial genome (Malpighiales) are potentially functional because they are transcribed (Xi et al., 2013). Relaxed transcription is known to occur in plant mitochondria (Brandt et al., 1993; Holec et al., 2006) and therefore the presence of a transcript does not confirm functionality. Many of the intact foreign mitochondrial genes in Geranium are unlikely to be functional, but additional analyses are needed to verify functionality of the foreign genes. Gene conversion between native and foreign copies of mitochondrial genes is a dynamic process that can generate chimeric genes and enhance genetic diversity in plant mitochondrial genomes. Previous studies have shown that gene conversion commonly occurs between foreign and native genes (Barkman et al., 2007; Hao et al., 2010; Mower et al., 2010; Hepburn et al., New Phytologist (2015) 208: 570–583 www.newphytologist.com

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2012). Gene conversion between small segments of foreign and native genes is evident in G. brycei cox2 (Fig. 3). The cox2 gene was transferred from Cuscuta via a single HGT event in common ancestor of the G. brycei and G. incanum clade, and gene conversion occurred only in G. brycei (Figs 2, 3). In addition to cox2, two mitochondrial versions of cox1, nad2 and rps3 that differ in intron content are present and may provide genetic diversity in Geranium mitochondrial genomes. Horizontal acquisition of the cox1 group I intron from fungi has been identified sporadically in plant mitochondrial genomes where complete genomic integration of the intron into the host intronless cox1 gene is seen (Cusimano et al., 2008; SanchezPuerta et al., 2008, 2011). Two alternative hypotheses pertain to the distribution of cox1i729 across plants: a single HGT event in a common ancestor followed by stochastic loss of the foreign intron vs widespread, independent horizontal gene/intron transfer. The two copies of cox1 present in G. brycei differ in intron content (Fig. 4b), which is unprecedented among sequenced plant mitochondrial genomes, supporting the notion of repeated, independent HGT. Furthermore the absence of coconversion sequences in the native copy makes it unlikely that an earlier integration and loss of cox1i729 had occurred in this species. Foreign MIPTs played a crucial role in identifying potential donors of foreign genes in Geranium. Plastid DNA can be transferred from one species into unrelated plants (Stegemann et al., 2012) and two examples have shown that mitochondrial DNAs harbor foreign MIPTs introduced by HGT (Woloszynska et al., 2004; Rice et al., 2013). Geranium brycei and G. incanum each contain at least 13 foreign MIPTs in their mitochondrial genomes. Phylogenetic analyses of plastid genes across angiosperms identified at least three potential donors in Cuscuta (Solanales), Acalyphoideae (Malpighiales) and Rubioideae (Gentianales), which are consistent with potential HGT donors of the foreign mitochondrial genes. The identification of Rubioideae as the likely plastid donor lineage could illuminate a possible donor of the foreign mitochondrial atp4 gene identified as sister to the Rhazya/Asclepias (Gentianales) clade (Fig S6a). There are two plausible explanations for the discovery of foreign MIPTs in the Geranium mitochondrial DNAs: (1) a direct HGT between plastids of donors and Geranium mitochondria, or (2) a HGT event from the mitochondrial genome of donors that already acquired plastid genes via IGT. The clustering of plastid and mitochondrial genes from the same donor in the G. brycei genome (Fig. 1) clearly supports the second explanation. Possible routes for plant-to-plant HGT include direct connections between the donor and host (Bock, 2010). Parasitism among plants has often provided a source of HGT substrates and many HGT events in Geranium likely occurred by direct plantto-plant transmission of DNA from parasitic plants, especially Cuscuta. Experimental studies have demonstrated that the parasite Cuscuta benefits by obtaining resources from host species, including both Geranium and Pelargonium (De Bock & Fer, 1992; Snyder et al., 2005). Both phylogenetic evidence (Figs 2, S6, S7) and the biogeographic distribution of donors and recipients support this scenario. For example, G. brycei and G. incanum are native to southern Africa (Aedo et al., 1998) and C. planiflora, New Phytologist (2015) 208: 570–583 www.newphytologist.com

which was identified as the likely gene donor by phylogenetic analysis (Fig. 2g), is also from southern Africa (Welman, 2003). The foreign copy of nad6 of G. traversii, which is native to New Zealand (Aedo et al., 1998), is grouped with Bartsia (Orobanchaceae, Fig. 2c) and some members of Orobanchaceae are distributed in New Zealand (Mabberley, 2008). Donor identification for HGT of nad6 requires further phylogenetic analysis using a member of Orobanchaceae found in New Zealand (probably including Australian species) to better understand the evolutionary history of this transfer. Illegitimate pollination, a proposed mechanism for HGT of the cox1 intron (reviewed in Mower et al., 2012a), could account for the HGT from Acalyphoideae (Ricinus) into Geranium mitochondrial DNAs. This study provides evidence for numerous HGT events in Geranium mitochondrial DNAs, indicating that the evolutionary history of mitochondrial genomes in this genus has been greatly impacted by the uptake of foreign DNA. The identification of gene conversion between foreign and native mitochondrial genes suggests that HGT plays an important role in generating mitochondrial genetic diversity in Geranium. Furthermore, the evolutionary progression of Geranium by HGT probably involves other genetic material (e.g. nuclear sequences) and other pathways (e.g. gene transfer into nuclear genome). A full understanding of the evolutionary history of HGT among Geranium genomes requires complete sequences for mitochondrial and nuclear genomes. The considerable number of HGT and IGT events in Geranium also suggests coordination of these two types of events but the evolutionary forces driving these phenomena are not yet clear. To better address this fundamental question about coordination between HGT with IGT in generating mitochondrial genome diversification in plants, additional genome sequences from other species with reduced gene content are needed to investigate the fate of the transferred genes.

Acknowledgements Support was provided by the National Science Foundation (IOS1027259 to R.J.K., T.A.R. and J.P.M.) and from Vice President for Educational Affairs Prof. Dr Abdulrahman O. Alyoubi (King Abdulaziz University Jeddah, Saudi Arabia to S.P. and J.S.). The authors thank Sasa Stefanovic for providing Cuscuta gronovii DNA, Katya Romoleroux for assistance with collection of Bartsia pecularoides and Robin Parer at www.Geraniaceae.com for providing living material of Geraniaceae species. We also thank the Genome Sequencing and Analysis Facility at the University of Texas at Austin for performing the Illumina sequencing, the Texas Advanced Computing Center (TACC) at the University of Texas at Austin for access to supercomputers and two anonymous reviewers for comments on an earlier version of the manuscript.

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Table S2 Information about phylogenetic analyses for horizontal gene transfer (HGT) of mitochondrial genes

Supporting Information Additional supporting information may be found in the online version of this article.

Table S3 Information about phylogenetic analyses for horizontal gene transfer (HGT) of plastid genes

Fig. S1 Mitochondrial genome map of Geranium maderense. Fig. S2 Mitochondrial protein-coding gene content in Geranium and related species. Fig. S3 Mitochondrial intron content in Geranium and related species. Fig. S4 Schematic diagram of split gene transfer of rpl2 from the mitochondrial genome to the nucleus.

Table S4 Information about phylogenetic analysis and divergence time Table S5 Status of mitochondrial genome assembly of Geranium and related species of Geraniaceae and Geraniales Table S6 Predicted RNA editing in 26 or 27 protein-coding genes for mitochondrial DNAs of 17 species of Geranium and Arabidopsis thaliana

Fig. S5 Phylogenetic analyses of native genes that do not have any additional copies in Geranium mitochondrial genomes.

Table S7 Summary of mitochondrial genes transferred to the nucleus in three species of Geranium and species from three other genera of Geraniaceae

Fig. S6 Phylogenetic evidence for horizontal gene transfer (HGT) of mitochondrial DNAs in Geranium mitochondrial genomes.

Table S8 Comparison of native and foreign mitochondrial genes

Fig. S7 Maximum-likelihood analyses across angiosperms of the 13 foreign mitochondrial DNA of plastid origin (MIPTs) in Geranium mitochondrial genomes. Fig. S8 Chronogram of Geraniales divergence times. Table S1 Information Geraniales

about

mitochondrial

genes

from

Table S9 Coverage for the contigs surrounding nonidentical mitochondrial genes and native genes Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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